A virus particle detection method based on cell membrane chromatography technology

Abstract

The application discloses a virus particle detection method based on a cell membrane chromatography technology and belongs to the field of virus detection methods and comprises the following steps: collecting virus particles in air aerosol, uniformly distributing the virus particles in an absorption liquid, then conveying the absorption liquid to an ACE2-CMC cell membrane chromatography column, conveying the eluate to a detector after elution, and realizing detection of the virus particles. The application realizes detection of the virus particles by collecting the virus particles in the air aerosol, uniformly distributing the virus particles in the absorption liquid, then detecting the eluate through the ACE2-CMC cell membrane chromatography column, is more convenient and faster than the RT-PCR method, and can complete the elution and detection process within 10 minutes without a strict laboratory enzyme-free environment. The method can realize continuous online detection of virus particles in environmental gas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus detection methods, and particularly relates to a virus particle detection method based on cell membrane chromatography technology. BACKGROUND

[0002] The most commonly used SARS-CoV-2 detection is molecular detection and antigen detection. Among them, molecular detection such as reverse transcription polymerase chain reaction (RT-PCR) detects the virus genetic material and is sensitive and reliable, and is the gold standard for detecting SARS-CoV-2. However, it also has some shortcomings, such as the need for enzyme-free operation in a molecular laboratory, tedious steps and time-consuming. Another method is antigen detection, which directly detects SARS-CoV-2 antigen, has low cost and fast detection speed, and patients can perform self-detection at home. However, the accuracy and sensitivity of antigen detection are poor at low viral load, which can easily lead to high false negative rate. Therefore, there is an urgent need for a rapid, high-sensitivity and low-cost virus detection technology to achieve rapid point-of-care testing.

[0003] Since SARS-CoV-2 can be transmitted through aerosols, it is necessary to study the detection of viral aerosols in the environment. However, there is currently a lack of rapid and effective detection methods. On the other hand, the continuous variation of the virus makes it more likely to escape immunity, so host-targeted drug screening can be used as a complementary strategy. Membrane protein angiotensin-converting enzyme 2 (ACE2) is a host receptor for SARS-CoV-2, and the construction of an ACE2 biosensor provides an effective way for virus recognition. Since many variants of SARS-CoV-2 exhibit strong binding to ACE2, the immobilization of ACE2 is expected to be suitable for SARS-CoV-2 variants with different pathogenicity.

[0004] Cell membrane chromatography (CMC) is a high-efficiency bioaffinity chromatography technology that immobilizes membrane proteins on the chromatographic stationary phase, has the dual advantages of chromatographic separation and biological recognition, and is easy to customize the stationary phase according to the target analyte, and is widely used in the field of ligand-receptor interaction analysis. Therefore, ACE2 / CMSP can also be used to bind to the spike protein to develop a convenient virus antigen detection method. So far, small-pore silica gel As a carrier, this carrier is suitable for the analysis of small molecule compounds, and the molecular weight and particle size of virus particles are large, which cannot enter the pores and are used for complex biological samples, which can easily cause excessive column pressure and cause chromatographic column blockage. Therefore, the application of cell membrane chromatography technology in the field of biological macromolecules is limited.

[0005] So far, there is no report on virus particle detection based on cell membrane chromatography technology, and cell membrane chromatography technology has not been developed and applied in the field of virus detection. SUMMARY

[0006] In order to overcome the shortcomings of the existing virus detection technology, the purpose of the present application is to provide a virus particle detection method based on cell membrane chromatography technology, which is simple to operate and can realize continuous online detection of virus particles and effective detection of virus aerosols in the environment.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] A virus particle detection method based on cell membrane chromatography technology, comprising the following steps:

[0009] Collecting virus particles in air aerosols, uniformly distributing the virus particles in the absorption liquid, then conveying the absorption liquid to the ACE2-CMC cell membrane chromatography column, after elution, conveying the eluate to the detector to realize detection of the virus particles.

[0010] Further, collecting virus particles in air aerosols, then uniformly distributing the virus particles in the absorption liquid, comprising the following steps: forming a negative pressure in the absorption pool of the liquid impact biological aerosol sampler by the air pump, air enters the gas collector under the negative pressure of the liquid impact biological aerosol sampler, and the virus particles are uniformly distributed in the absorption liquid.

[0011] Further, the air flow rate is 5 L / min.

[0012] Further, the absorption liquid enters the ACE2-CMC cell membrane chromatography column at a flow rate of 0.1 mL / min.

[0013] Further, during elution, elution is carried out at a flow rate of 0.2 mL / min.

[0014] Further, the ACE2-CMC cell membrane chromatography column is prepared by the following process: the HEK-293T-ACE2 cell membrane solution is immersed into the bis(vinylsulfonyl)methane modified silica gel, after incubation, the supernatant is discarded, and the stationary phase suspension is obtained; the stationary phase suspension is packed into the column core by wet packing, and the ACE2-CMC cell membrane chromatography column is obtained.

[0015] Further, the bis(vinylsulfonyl)methane modified silica gel is prepared by the following process: after drying and activation, the macroporous silica gel is added into acetonitrile together with bis(vinylsulfonyl)methane and 4-dimethylaminopyridine, and stirred to react, to obtain the bis(vinylsulfonyl)methane modified silica gel.

[0016] Further,

[0017] Further, the ratio of the use amount of super-large-pore silica gel, bis (vinyl sulfone group) methane and 4-dimethylamino pyridine is 0.1g:100.12mg:2mmol.

[0018] Further, the temperature of the dry activation is 105 DEG C, and the time is 30min; the stirring reaction time is 12h.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application realizes the detection of virus particles by collecting virus particles in air aerosol, uniformly distributing the virus particles in the absorption liquid, and then detecting the eluent by the ACE2-CMC cell membrane chromatographic column, which is more convenient and faster than the RT-PCR method, and the elution and detection process can be completed within 10min without a strict laboratory enzyme-free environment. 3 The detection limit is 3.8x10 3 VP / mL, which is higher than the antigen test paper method for detecting virus antigens. The method can be used not only for detecting human-derived biological liquid samples, but also for continuous real-time monitoring of environmental aerosol or water samples by simply changing the sampling method. The method can be used as a general platform for detecting virus particles, and only needs to prepare a chromatographic column with a fixed corresponding virus receptor for a specific virus to realize the detection of multiple viruses.

[0021] Further, the present application uses super-large-pore silica gel to replace conventional pore size silica gel which can make virus particles (80-120nm) enter the internal pore size of the silica gel particles, enhance the interaction between the virus biological macromolecule analyte and the stationary phase, and at the same time, due to the larger internal pore size, the overall column pressure after loading is lower, and the rapid analysis of complex biological source samples can be realized. Compared with the currently used cell membrane chromatography stationary phase which is suitable for small molecule compound analysis, the present application first introduces super-large-pore silica gel, which expands the application range of the cell membrane chromatography stationary phase to the field of virus biological macromolecules.

[0022] Further, in the present application, the absorption liquid in the absorption cell is pumped to the cell membrane chromatographic column, i.e. the ACE2-CMC cell membrane chromatographic column, and the virus particles are specifically combined with the virus receptors on the stationary phase and are captured, and the remaining components do not have a retention effect because they do not have obvious specific interaction with the virus receptors. After continuous circulation, the number of viruses combined on the cell membrane chromatographic column gradually increases with time, which plays a role in enrichment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 This is a diagram illustrating the process of virus detection using cell membrane chromatography.

[0024] Figure 2 Scanning electron microscope (SEM) images of ACE2-HEK293T cell membranes before and after bonding to ultra-large pore silica gel; where (a) is a scanning electron microscope image of BVS-modified silica gel, and (b) is a scanning electron microscope image of the silica gel stationary phase after ACE2 bonding.

[0025] Figure 3 Immunofluorescence images showing the specific binding of ACE2 cell membrane chromatographic stationary phase to SARS-CoV-2 spike pseudovirus;

[0026] Figure 4 To identify and detect SARS-CoV-2 spike pseudovirus, ultraviolet absorption spectra, viral signal graphs at different concentrations, and viral detection calibration curves were obtained; where (a) is the ultraviolet absorption spectrum, (b) is the viral signal graph at different concentrations, and (c) is the viral detection calibration curve.

[0027] In the diagram, 1 is the bioaerosol sampler, 2 is the absorption cell, 3 is the first high-pressure infusion pump, 4 is the cell membrane chromatography column, 5 is the second high-pressure infusion pump, 6 is the eluent, and 7 is the PDA detector. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] The present invention will now be described in further detail with reference to the accompanying drawings.

[0030] In this invention, a membrane protein receptor with specific high affinity for the target virus is immobilized on a cell membrane chromatography column. This invention is illustrated using the detection of the novel coronavirus as an example.

[0031] This invention provides a method for detecting virus particles based on cell membrane chromatography technology. First, ultra-large pore silica gel is prepared. The cell membrane chromatographic column bonding the virus specific membrane receptor is used as a recognition and complement unit of virus particles, and then a pipeline is connected with a sampler, a high-pressure infusion pump, the cell membrane chromatographic column and a detector, a detection signal is output by a computer, virus receptors on a stationary phase are specifically combined with virus surface proteins, so that viruses in a complex sample system are captured and separated, and it is a virus particle rapid detection method which integrates gas collection, virus enrichment, elution and detection.

[0032] In the application, the silica gel with super-large aperture is used as a stationary phase carrier, so that virus particles to be detected enter the internal aperture of the silica gel stationary phase, the contact and combination with the receptors on the stationary phase are enhanced, and meanwhile the characteristics of the large aperture also ensure low column pressure, so as to facilitate the analysis of samples with complex components such as virus absorption liquid, blood samples and the like.

[0033] The silicon hydroxyl on the surface of the super-large pore silica gel is modified by bis (vinyl sulfone) methane (BVS), specifically, 4-dimethylaminopyridine (DMAP) is used as a catalyst, and the Michael addition reaction of BVS and the silicon hydroxyl occurs.

[0034] The membrane receptor is obtained by extraction and purification of the His-tag labeled target membrane protein by styrene-maleic acid copolymer (SMA).

[0035] The specific steps of the application are as follows:

[0036] 1) Preparation of a cell membrane chromatographic column for recognizing biological macromolecules

[0037] a) BVS functional modification of silica gel

[0038] 0.1g of super-large pore silica gel is placed in a 105℃ drying oven for activation for 30min, then 100.12mg of bis (vinyl sulfone) methane BVS and 2.44mg of 4-dimethylaminopyridine (DMAP) are added into 10mL of dried acetonitrile, and the reaction is stirred at room temperature for 12h, after the reaction is completed, the silica gel is washed with acetonitrile and water, and is dried in an 85℃ oven, to obtain the BVS modified silica gel.

[0039] b) Cell membrane receptor immobilization

[0040] The ACE2-HEK293T (i.e. HEK-293T-ACE2) cell membrane solution extracted by ultrasonic crushing and differential centrifugation is mixed with the BVS modified silica gel, the cell membrane solution is immersed in the cell membrane solution, and the mixture is incubated on a shaker at room temperature for 12h, after the incubation is completed, the supernatant is discarded by centrifugation at 5000r for 5min, and the silica gel is resuspended with PBS, to obtain a stationary phase suspension.

[0041] Reference Figure 2Under the scanning electron microscope, the supermacroporous silica gel showed connected pores. After the incubation reaction with the cell membrane solution, a layer of membrane-like substance was visible on the surface of the silica gel, indicating that the cell membrane was successfully fixed on the surface and pores of the silica gel.

[0042] c) Wet packing

[0043] Take 1 mL of the stationary phase suspension and pack it into a 2.0 x 10 mm (I.D. x L) column core by wet packing, to obtain an ACE2-CMC cell membrane chromatographic column.

[0044] Referring to Figure 3 The prepared ACE2 cell membrane stationary phase was verified by immunofluorescence for virus binding. The results showed that ACE2 (green fluorescent antibody labeled) on the surface of the silica gel and S protein (red fluorescent antibody labeled) of the SARS-CoV-2 pseudovirus particles showed good colocalization. There was no such colocalization in the solvent control group and the negative control group, verifying that the sACE2 on the cell membrane chromatographic stationary phase had good specific binding with the S protein of SARS-CoV-2.

[0045] 2) Construction of detection platform

[0046] Referring to Figure 1 Connect the detection modules as shown in the figure, wherein 5 mL of 1 mmol / L phosphate buffer (pH 7.0) is added to the absorption cell as the absorption liquid for virus particle aerosol, and the flow rate is 0.1 mL / min. The first high-pressure infusion pump 3 is connected to the absorption cell and the cell membrane chromatographic column. The flow cell of the PDA detector 7 is connected after the column, and then connected back to the absorption cell. The second high-pressure infusion pump 5 is connected to the eluent (10 mmol / L Na2PH4) and the cell membrane chromatographic column 4. The flow rate of the eluent is 0.2 mL / min.

[0047] 3) Detection of virus particles

[0048] a) Sampling

[0049] The liquid impact biological aerosol sampler 1 is used to collect virus particles in the air aerosol. First, a negative pressure is formed in the absorption cell 2 of the liquid impact biological aerosol sampler 1 by operating the air pump, and air enters the gas collector under the negative pressure of the liquid impact biological aerosol sampler 1 at a flow rate of 5 L / min. Virus particles are uniformly distributed in the absorption liquid under the action of airflow impact and gas-liquid flow. At the same time, the porous glass plate promotes the formation of a large number of small gas bubbles, increases the contact area between the gas and the absorption liquid, and improves the virus absorption efficiency.

[0050] b) Virus identification and enrichment

[0051] The absorption solution is pumped into the ACE2-CMC cell membrane chromatographic column at a rate of 0.1 mL / min by the first high-pressure infusion pump 3. The coronavirus is specifically bound to the ACE2 on the chromatographic column through the Spike protein on the surface, and the virus is enriched in the ACE2-CMC cell membrane chromatographic column. The absorption solution is then circulated into the absorption pool.

[0052] c) Virus elution

[0053] After the virus is enriched on the ACE2-CMC cell membrane chromatographic column for a period of time, the second high-pressure infusion pump 5 pumps the eluent out at a rate of 0.2 mL / min to flush the column, elutes the enriched virus, and transports the virus in a high concentration state to the detector.

[0054] When the binding sites on the cell membrane chromatographic column approach saturation, the mobile phase is switched to the eluent 6 with stronger elution ability, which weakens the interaction between the virus and the receptor, thereby eluting the virus particles and flowing to the detector along the liquid pipeline.

[0055] The absorption solution in the absorption pool is pumped to the cell membrane chromatographic column, i.e., the ACE2-CMC cell membrane chromatographic column, by the high-pressure infusion pump. The virus particles are specifically bound to the virus receptors on the stationary phase and are captured, and the remaining components do not have a retention effect because they do not have significant specific interaction with the virus receptors, and continue to circulate back to the absorption pool. Through continuous circulation, the number of viruses bound on the cell membrane chromatographic column gradually increases over time, playing an enrichment role. The coronavirus is specifically bound to the ACE2 on the chromatographic column through the Spike protein on the surface, and the virus is enriched in the ACE2-CMC cell membrane chromatographic column. The absorption solution can be circulated into the absorption pool.

[0056] d) Detection

[0057] The eluent is detected by the UV detector, i.e., the PDA detector 7, and quantified according to the characteristic retention signal intensity of the virus.

[0058] The virus eluted from the cell membrane chromatographic column has a characteristic retention time, and the remaining components do not have a retention and peak at the solvent peak position. The virus particles have a characteristic retention time, and the characteristic peak of the virus is detected by the UV detector, and the peak area or peak height is used for quantitative detection.

[0059] Reference Figure 4 The ultraviolet absorption spectrum of SARS-CoV-2 Spike pseudovirus was scanned by ultraviolet spectrophotometer (a) in Figure 4 It was found that SARS-CoV-2 Spike pseudovirus had characteristic absorption near 214 nm and 280 nm, with stronger absorption at 214 nm, so 214 nm was used as the detection wavelength. Then different titers of SARS-CoV-2 Spike pseudovirus were injected into the ACE2-CMC column. As Figure 4As shown in (b), using high concentration phosphate buffer as elution buffer, SARS-CoV-2 spike pseudovirus can produce obvious retention peak on ACE2-CMC model, and the signal intensity of virus characteristic peak increases with the increase of virus concentration. The calibration curve shows that the improved method can identify virus concentration as low as 80 VP µL-1 Figure 4 As shown in (c), the limit of detection (LOD) is estimated to be 3.8 x 10 3 VP / mL, defined as 3 times the standard deviation.

[0060] In the present application, the carrier of the cell membrane chromatography stationary phase is silica gel with super-large pore size, which effectively enhances the mutual recognition efficiency of biological macromolecules such as virus particles and membrane receptors, and the column pressure is lower, which is suitable for the analysis of complex biological samples.

[0061] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for detecting virus particles based on a cell-membrane chromatography technique, characterized by, The method comprises the following steps: The virus particles in the air aerosol are collected, the virus particles are uniformly distributed in the absorption liquid, and then the absorption liquid is delivered to the ACE2-CMC cell membrane chromatographic column, after elution, the eluate is delivered to the detector, and detection of the virus particles is realized; The ACE2-CMC cell membrane chromatographic column is prepared by the following process: the HEK-293T-ACE2 cell membrane solution is immersed into the bis(vinylsulfonyl)methane modified silica gel, the supernatant is discarded after incubation, and a stationary phase suspension is obtained; the stationary phase suspension is packed into the column core by wet packing, and the ACE2-CMC cell membrane chromatographic column is obtained; The bis(vinylsulfonyl)methane modified silica gel is prepared by the following process: after the macroporous silica gel is dried and activated, the bis(vinylsulfonyl)methane and 4-dimethylaminopyridine are added to acetonitrile, and stirring reaction is performed to obtain the bis(vinylsulfonyl)methane modified silica gel. The pore size of the macroporous silica gel is greater than or equal to 1500 angstroms.

2. The method of claim 1, wherein the cell membrane chromatography-based virus particle detection method is characterized by, The virus particles in the air aerosol are collected, and then the virus particles are uniformly distributed in the absorption liquid, comprising the following steps: a negative pressure is formed in the absorption pool of the liquid impact biological aerosol sampler by a gas pump, air enters the gas collector under the action of the negative pressure of the liquid impact biological aerosol sampler, and the virus particles are uniformly distributed in the absorption liquid.

3. The method of claim 2, wherein the cell membrane chromatography-based virus particle detection method is characterized by, The air flow rate is 5 L / min.

4. The method of claim 1, wherein the cell membrane chromatography-based virus particle detection method is characterized by, The absorption liquid enters the ACE2-CMC cell membrane chromatographic column at a flow rate of 0.1 mL / min.

5. The method of claim 1, wherein the cell membrane chromatography-based virus particle detection method is characterized by, During elution, elution is performed at a flow rate of 0.2 mL / min.

6. The method of claim 1, wherein the cell membrane chromatography-based virus particle detection method is characterized by, The amount ratio of the macroporous silica gel, the bis(vinylsulfonyl)methane and the 4-dimethylaminopyridine is 0.1 g:100.12 mg:2 mmol.

7. The method of claim 1, wherein the cell membrane chromatography-based virus particle detection method is characterized by, The drying and activation temperature is 105 DEG C, and the time is 30 min; the stirring reaction time is 12 h.

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